Demethylation induces <i>Oxtr</i> expression in 4T1 cells.

<p><b>A</b>) In GT1-7 cells, which normally show high <i>Oxtr</i> mRNA levels and low methylation, A two-day treatment with 5-AzaC caused a slight reduction in <i>Oxtr</i> mRNA levels <b>B</b>)Conversely, treatment of 4T1 cells with 5-AzaC caused a robust dose-dependent increase in <i>Oxtr</i> mRNA levels.. <b>C</b>) 5-AzaC treatment (2 µM) of 4T1 cells caused a significant reduction in the methylation of the <i>Oxtr</i> promoter (compared to vehicle samples, also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056869#pone-0056869-g002" target="_blank">Fig. 2B</a>) across all seven CpG sites of the amplicon. <b>Statistics</b>: <b>A</b>) One-way ANOVA F₍₃₎ = 90.1, <i>p</i><0.001, * differ from vehicle-treated, † differ from 0.5 µM, <i>p</i><0.05, n = 5. <b>B</b>) One-way ANOVA F₍₃₎ = 39.6, <i>p</i><0.001, * differ from vehicle-treated, <i>p</i><0.01, n = 5.</p

PCR primer sets.

<p>T_A, Temperatures and time applied for annealing.</p

Additional file 1 of Impacts of inflammatory cytokines on depression: a cohort study

Supplementary Material

TEM analysis of glycogen synthesis and the formation of glycogen bodies mediated by BMP-4 and GSK-3 inhibition in hESCs.

<p>(<b>A</b>) Glycogen body formation in untreated H1 cells (control). (<b>B</b>) Glycogen body formation in BMP-4 treated H1 cells. (<b>C</b>) Glycogen body formation in GSK3i (CHIR99021)-treated H1cells. The asterisk sign indicates a glycogen defect region in the glycogen body, which likely resulted from dissociation of glycogen aggregates when the specimens were floating in solution during sample preparation. Additional TEM micrographs are available in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.s002" target="_blank">S2 Fig</a>. (<b>D</b>) Box-and-Whisker plots of glycogen to cell ratios (with 10–90% percentile) in hESC control (n = 49 cells), BMP-4 treated cells (n = 29), and GSK3i treated cells (n = 22). The statistics included both H1 and H9 cells to increase statistical power. All hESC (i.e., H1 and H9) cells were grown as a non-colony type monolayer (mc) on 2.5% BD Matrigel in meTeSR1 medium and then treated with 100 ng/mL of BMP-4 and 3 μM GSK3i for 48 hours. The red-colored arrowheads indicate the formation of glycogen bodies (GB) in the cytoplasm of the cell. The plus signs (+) in the plots indicate the location of mean values. Abbreviations: GB, glycogen bodies with defined boundaries; Nu, the nucleus of the cells. Scale bars in (A, B) represent 2 μm; and scale bars in (C) represent 1 μm.</p

Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).

<p>Oct-4 expression in H1 control cells (A), H1 cells treated with 100 ng/mL of BMP-4 (B), and H1 cells treated with 3 μM GSK3i (CHIR99021) for 48 hours (C). The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (<b>D</b> and <b>E</b>) Box-and-Whisker plots of Oct-4 expression (with 5–95% percentile) in both H1 and BC1 cells under the indicated treatments. Control 2 (Cont2) in D is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of the mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars represent 50 μm.</p

<i>Oxtr</i> mRNA levels correlate with <i>Oxtr</i> promoter methylation in cell lines.

<p><b>A</b>) The relative mRNA levels in GT1-7 are significantly higher than in 4T1 cells. <b>B</b>) Methylation of the seven CpG sites is higher in 4T1 cells than in GT1-7 cells. Each row represents a single clone and each column represents one of the seven CpG sites. The total percentage of methylation was calculated from the fraction of black spots (methylated CpG sites). <b>C</b>) Representative gel showing greater ERK phosphorylation in GT1-7 cells stimulated with 1 µM OT for 10 min compared vehicle-treated cells Graph is summary of three independent experiments. <b>D</b>) Quantities of <i>Oxtr</i> mRNA in GT1-7 cells following 24 h treatment with 1 µM OT. An OT-stimulated increase in <i>Oxtr</i> mRNA was documented in three independent experiments. <b>Statistics</b> (t-test): <b>A</b>) *<i>p</i><0.001, n = 4. <b>C</b>) *<i>p</i><0.05, n = 3. <b>D</b>) *<i>p</i><0.01 n = 3.</p

Methylation of specific CpG sites in the <i>Oxtr</i> minimal promoter inhibits transcription.

<p><b>A</b>) A schematic depiction of the distinct <i>Oxtr</i> promoter/<i>EYFP</i> constructs used; An <i>EYFP</i> gene was coupled to a minimal promoter (positions −1417 to +46) of the mouse <i>Oxtr</i> gene (<i>Unmodified</i>). This construct was modified by a C to A mutation at CpG sites 1 (<i>Mut 1</i>) or site 7 (<i>Mut 7</i>) or by deleting the ∼400 bp amplicon region (<i>Del</i>). <b>B,C</b>) EYFP mean fluorescence intensity measured in GT1-7 cells that were transfected with either (<b>B</b>) untraeatd or (<b>C</b>) methyltransferase-treated <i>Oxtr</i> promoter/<i>EYFP</i> constructs. No significant differences were found among the untreated plasmids whereas a highly significant difference was found among the methyltransferase-treated plasmids. <b>D</b>) A comparison between the treatedand untreated versions of all plasmids. Highly significant differences were found only in the cases of the <i>Unmodified</i> and <i>Mut 1</i> plasmids. <b>Statistics</b>: <b>B</b>) one-way ANOVA F₍₃₎ = 2.32, <i>p</i>>0.1. <b>C</b>) - one-way ANOVA F₍₃₎ = 9.4, <i>p</i><0.01, * differ from <i>Unmodified</i>, <i>p</i><0.05, n = 5. <b>D</b>) t-test, *<i>p</i><0.05/4.</p

2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.

<p>(<b>A</b>) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (<b>B</b>) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images from 2-NBDG alone. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (<b>C</b>) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g005" target="_blank">Fig 5</a>. (<b>D</b>) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (<b>E</b>, <b>F</b>) Quantitative analysis of 2-NBDG retention and glycogen labeling by measuring mean fluorescence intensity (FI, arbitrary units) from at least 4 random colonies (E) and by counting 2-NBDG loci (F). Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Scale bars represent 100 μm.</p

Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).

<p>A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (<b>A)</b> In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (<b>B</b>) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (<b>C</b>) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (<b>D</b>) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (<b>E</b>) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (<b>F</b>) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (<b>G</b>) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (<b>H</b>) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.</p

The general structure of the mouse <i>Oxtr</i> gene and its promoter.

<p><b>A</b>) A schematic depiction of the mouse <i>Oxtr</i> gene, including the CpG island that extends from the promoter into the coding region at the third exon. <b>B</b>) A schematic depiction of the EREs and SP1 binding sites in the minimal gene promoter, and the relative location of the PCR amplicon between positions −956 and −541. <b>C</b>) The sequence of the PCR amplicon, including the positions of the seven CpG sites. The ERE harboring CpG site 1 and the SP1 binding sequence harboring CpG site 7 are underlined.</p